The chronic respiratory infections with mucoid Pseudomonas aeruginosa are the major cause of high mortality and morbidity in cystic fibrosis (CF). The specific expression patterns of virulence factors (e.g. mucoidy and reduced protease levels) play a significant role in the persistence of Pseudomonas infections in CF. Mucoidy, caused by the overproduction of the exopolysaccharide alginate, is regulated at the transcriptional level via two-component signal transduction systems (including the algR gene) and a unique histone-like element (AlgP). Overexpression of alginate genes is promoted by muc mutations and is responsive to a variety of environmental stimuli (e.g. nitrogen, phosphate, sulfate limitations and osmolarity changes). This proposal is aimed at determining the mechanisms of the dual control of mucoidy by signal transduction systems and the histone-like element AlgP. A possible integration of the regulatory inputs from signal transduction systems with the general state of bacterial nucleoid and its role in the control of mucoidy will be examined. The specific objectives are: (i) To purify the algR gene product and analyze its interactions with the algD promoter; algD, encodes a key alginate biosynthetic enzyme and undergoes strong transcriptional activation in mucoid cells. These events play a pivotal role in the establishment of mucoidy. (ii) To identify and characterize the second (sensory/kinase) component interacting with AlgR and study the signal transduction processes. Also, a possible contribution of different signal transduction systems to the regulation of alginate synthesis will be examined. (iii) Two cloned muc genes and mutations that result in the overproduction of alginate and are conducive to mucoid phenotype will be characterized. Interactions of these loci wit the regulation by histone-like elements and signal transduction systems will be investigated. (iv) The function of AlgP, a histone-like element will be determined. Its role in the control of the local promoter conformations will be assessed. The entire C-terminal half of AlgP resembles histones H1 from eukaryotes. The multiple 75 bp, 12bp, and 3 bp direct repeats encoding this region of AlgP are the basis for DNA rearrangements resulting in clonal variations of this region. Rearrangements in algP are frequently associated with a loss of mucoidy. The possible role of this novel switch in CF infections will be analyzed using polymerase chain reaction in longitudinal studies on specimens from CF patients. The proposed studies will permit understanding of the cooperation of regulatory networks in adaptation processes in bacterial pathogenesis. Analyses of the signal transduction systems controlling mucoidy in P. aeruginosa could lead into a future design of inhibitors of these processes and improvement of antimicrobial therapies in CF.